Hybrid mass spectrometer

ABSTRACT

A data independent acquisition method of mass spectrometry for analyzing a sample within a mass range of interest as it elutes from a chromatography system. The method comprises selecting precursor ions within a mass range of interest to be analyzed, performing at least one MS1 scan of the precursor ions using a Fourier Transform mass analyser and performing a set of MS2 scans by segmenting the precursor ions into a plurality of precursor mass segments, each precursor mass segment having a mass range of no greater than 5 amu, and for each precursor mass segment fragmenting the precursor ions within that precursor mass segment and performing an MS2 scan of the fragmented ions.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority benefit under 35 U.S.C. § 119 toEuropean Patent Application No. 17174365.1, filed on Jun. 2, 2017, thedisclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to data independent analysis (DIA) oforganic and in particular biological samples such as proteins, peptides,metabolites, lipids and the like. In particular, it relates to a highresolution data independent identification and quantification techniquewith applications in proteomics, metabolomics, lipidomics and so forth.

BACKGROUND OF THE INVENTION

Mass spectrometry is a long established technique for identification andquantitation of often complex mixtures of large organic molecules. Inrecent years, techniques have been developed that allow analysis of awide range of both biological and non-biological materials, withapplication across the fields of law enforcement (e.g. identification ofdrugs and explosives materials), environmental, scientific research, andbiology (e.g. in proteomics, the study of simple and complex mixtures ofproteins, with applications in drug discovery, disease identificationand so forth).

Proteins, comprising large numbers of amino acids, are typically ofsignificant molecular weight. Thus accurate identification andquantitation of the protein by direct mass spectrometric measurement ischallenging. It is thus well known to carry out fragmentation of theprecursor sample material. A variety of fragmentation techniques areknown, which may result in the generation of different fragment ionsfrom the precursor ions. Moreover, the fragmentation mechanism may beaffected by different applied fragmentation energies.

Analysis of samples can broadly be separated into data independentanalysis/acquisition (DIA) and data dependent analysis/acquisition (DDA)techniques. DIA seeks to determine what is present in a sample ofpotentially unknown identity. To determine the molecular structure ofsample molecules, a mass spectrometer is first used to mass analyse allsample ions (precursor ions) within a selected window of mass to chargeratio (m/z). Such a scan is often denoted as an MS1 scan. The selectedsample ions are then fragmented and the resulting fragments aresubsequently mass analyzed across the selected m/z range. The scan ofthe fragmented ions is often denoted as an MS2 scan.

DDA by contrast, seeks to confirm that one or more species is/arepresent in a given sample. Methods of DDA identify a fixed number ofprecursor ion species, and select and analyze those via massspectrometry. The determination of which precursor ion species are ofinterest in DDA may be based upon intensity ranking (for example, thetop ten most abundant species as observed by peaks in a MS1 spectrum”),or by defining an “inclusion list” of precursor mass spectral peaks (forexample by user selection), from which MS2 spectra are always acquiredregardless of the intensity ranking of the peak in the MS1 massspectrum. Still otherwise, an “exclusion list” of peaks in MS1 can bedefined, for example by a user, based e.g. on prior knowledge of theexpected sample contents.

DIA avoids the decisions necessary in DDA, by simply dividing the massrange of interest (typically user defined) into segments and obtainingMS2 spectra for each segment. With DIA, the acquisition of an MS1precursor spectrum becomes more or less optional, since the parametersof the selection window for the sample ions carries information aboutthe range of possible sample ions within that window.

Early DIA techniques were disclosed in patent applications by MicromassUK Ltd and Waters Technologies Corporation, in their so-called MS^(E)arrangements. The DIA techniques resulted from application of knowntriple quadrupole methods to quadrupole-TOF arrangements.

In U.S. Pat. No. 6,717,130, a technique is disclosed in which MS1 andMS2 are alternatively acquired by repeatedly switching the energy of thefragmentation cell. The technique relies upon separation of the samplemolecules through different elution times in a chromatographyenvironment. At the end of an experimental run, precursor and fragmentions are recognized by comparing the mass spectra in the two differentfragmentation modes. Fragment ions are matched to particular precursorions on the basis of the closeness of fit of their elution times, sothat precursor ions can then be identified.

U.S. Pat. No. 6,982,414 discloses a development to the DIA technique inthe '130 patent described above. Here, the energy applied to thefragmentation cell is again repeatedly switched so as to obtain both MS1and MS2. However here MS1 and MS2 are obtained from both a first and asecond sample separately.

The mass spectra are then compared and further analysis is carried outwhere precursor ions in MS1 from each sample, or fragment ions in MS2from each sample, are expressed differently.

Finally U.S. Pat. No. 7,800,055 again employs switching between energylevels in the fragmentation chamber so as to generate MS1 and MS2 inalternating manner. Comparison of the chromatographic peak shape of theprecursor and fragment peaks is carried out to identify an associationbetween precursor and fragment (product) ions.

An alternative approach to DIA, known as “SWATH”, has been proposed invarious patents to DH Technologies Development Pte. Ltd.

In U.S. Pat. No. 8,809,770, a DIA data set is acquired such that thedata may subsequently be analyzed for a target substance. This contrastswith the idea of setting a target and then acquiring data only for thatpurpose. The method employs LC-MS and uses wide windows of precursorions (e.g. >10, >15, >20 amu) for MS2, allowing the whole precursorspace to be covered.

Again the '770 patent stresses the importance of retaining the fidelityof the chromatographic peaks in the MS2 spectrum, by appropriate settingof the windows. An MS1 spectrum is indicated to be optional.

As an example, the '770 patent describes a method—akin to singlereaction monitoring (SRM)—for evaluation of the MS2 data of a precursormass window as a function of retention time, and for subsequentcomparison with a reference spectrum library.

U.S. Pat. No. 8,809,772 employs isolation windows for the precursorions, of variable width, the width being dependent upon the precursormass. The method trades off analysis speed (for a wide window) andsensitivity/specificity (for a narrow window).

U.S. Pat. No. 9,343,276 addresses drawbacks with the methods disclosedin U.S. Pat. No. 8,809,770 by scoring extracted ion chromatogram (XIC)peak candidates based on various criteria, in a comparison between theXIC fragment peaks with the MS1 information, such as mass accuracy,charge state, isotopic state, known neutral losses and so forth.

A common aspect of the approach in the MS^(E) and SWATH techniquesdescribed above is that they seek to optimize measurements for good MS2time resolution. To obtain sufficient data points across the LC peak forgood quantitation, either a relatively wide precursor isolation window(24 Da)—as suggested in the MS^(E) patents discussed above—or a variablewidth precursor isolation window (the preferred approach for the SWATHpatents discussed above) needs to be employed.

The consequence is that the traditional database search—in which samplefragment spectra are compared against fragment spectra of known speciesin a library, as is the case with DDA—may not be well suited for DIAdata analysis. In the case of the SWATH technique, a large spectrallibrary must first be created (for the same or similar sample types) fortargeted extraction of MS2 chromatograms from convoluted spectra, whichis an expensive and time consuming task.

In “Evaluation of Data-independent Acquisition (DIA) Approaches forSpiked Peptides in HeLa Digest on Q-OT-qIT Mass Spectrometer”, Wei Zhanget al. (available athttp://tools.thermofisher.com/content/sfs/posters/PN-64122-Q-OT-qIT-ASMS2014-PN64122-EN.pdf)a method of DIA analysis using a tandem mass spectrometer is disclosed.In particular, an Orbitrap mass spectrometer is used to perform MS1scans of precursor ions, while a linear ion trap is used to perform MS2analysis of the fragmented ions using a mass window of 3 amu for eachMS2 scan. By using a narrow mass window, the resulting data can bedirectly used for database searching, realizing the integration of DDAand DIA methodologies.

SUMMARY OF THE INVENTION

For DIA methodologies performed on narrow chromatographic peaks of theorder of several seconds, one ideally needs to sample thechromatographic peak at least seven or better still at least ten timesover the full m/z range. This requirement constantly forces shorter andshorter DIA cycle times, currently 2-3 seconds is considered the upperlimit of allowed cycle time for applications in proteomics andmetabolomics. At the same time, the requirement for deeper coverage ofdifferent proteomes/metabolomes leads to bigger and bigger spectrallibraries being required for quantitation of the DIA data.

The present inventors have realized that one problem with existing DIAmethodologies is that large spectral libraries of fragment spectra arerequired for quantitation of fragment spectra in the MS2 domain. Thepresent inventors have realized that it would be desirable to move to alibrary free approach for DIA analysis of samples. In order to achievesuch library free analysis, the present inventors propose a method andapparatus that enables significantly higher specificity and speed ofDIA.

For example, a method of DIA analysis using a mass window of 3 amu foreach MS2 scan is discussed above. This relatively narrow mass window issimilar to the targeted mass windows used in a known DDA approach. Byusing relatively narrow mass windows for each of the MS2 scans, theresulting MS2 spectra may be analyzed using DDA type databases, ratherthan requiring full DIA-specific spectral libraries.

The present inventors have identified that one problem with using such anarrow mass window for each of the MS2 scans, is that the number ofscans required to build up a full range of MS2 scans for the mass rangeof interest increases. As a result, the time taken to perform a completeDIA analysis can become overly long, for example much longer than theduration of a chromatographic peak. If the DIA analysis is not completedwithin the duration of the chromatographic peak, the analysis may notprovide meaningful data, as some parts of the analysis will not measurethe sample peak.

Furthermore, another problem identified by the inventors with thisapproach, is that the relatively narrow mass windows for each MS2 scanresult in relatively few, if any, fragmenting ions reaching the detectorat a given time. Thus, reducing the mass selection window for each MS2scan results in a reduction in the mass accuracy and/or the sensitivityof the MS2 mass analyzer. This problem is exacerbated when attempting toperform a large number of MS2 scans at a sufficient frequency to fit allMS2 scans within the duration of a chromatographic peak.

The present invention seeks to address the shortcomings of existing DIAapproaches. In accordance with a first aspect of the present invention,there is provided a method of data independent analysis (DIA) inaccordance with claim 1. The invention also extends to a massspectrometer in accordance with claim 14.

In order to acquire data suitable for identification/quantitation ofprecursor ions using a DDA type database, the present invention acquiresMS1 and MS2 spectra utilizing high resolution, high mass accuracy massanalyzers. In particular, in the MS2 domain, the present inventionacquires MS2 spectra of narrow mass segments of less than 5 amu. In thisway, the MS2 spectra can be analyzed with DDA-type databases. Inparticular, it is important that the MS2 spectra are acquired using highmass accuracy, high resolution mass analyzers in order to identifyfragments with sufficient accuracy for the DDA-type databases to beapplicable.

Advantageously, by utilizing a Time of Flight (TOF) mass analyzer intandem with a FTMS mass analyzer, high mass accuracy scans may beperformed in both the MS1 and MS2 domains. Furthermore, the MS2 scansutilize a TOF mass analyzer which is preferably operated at a frequencyof at least 100 Hz in order to perform the plurality MS2 scans of theprecursor mass segments of less than 5 amu. By performing the pluralityof MS2 scans at such a frequency, MS2 spectra corresponding to narrowwindows of precursor ions may be acquired across the full mass range ofinterest within a cycle time that allows the DIA methodology to fullysample a chromatographic peak.

Importantly, MS1 data may be acquired across the full mass range ofinterest in tandem with the acquiring of the MS2 data. By utilizing aFTMS mass analyzer, high resolution high mass accuracy MS1 data may alsobe acquired to complement the MS2 data. Furthermore, by acquiring theMS1 data in tandem with the MS2 data, the total cycle time may bereduced compared to performing MS1 scans serially with the MS2 scans.

Preferably, the fragmented ions are accumulated prior to injection intothe time of flight mass analyzer. By accumulating fragmented ions for apredetermined time (duration) prior to mass analysis in the MS2 domain,the number of fragmented ions in each packet of ions is increased.Methods and apparatus for accumulation of ions are described below. Thismay lead, in preferred embodiments, to all fragmented ions beingutilized for MS2 scans, thereby increasing the duty cycle. As a time offlight mass analyzer is used to mass analyze each packet of fragmentedions, by increasing the number of ions in each packet, the intensity ofthe signal detected at a detector of the time of flight mass analyzer isincreased.

Preferably, the time of flight mass analyzer is operated at a frequencyof at least 100 Hz or at least: 150 Hz, 200 Hz, 250 Hz or 300 Hz. Byproviding a minimum operating frequency for the TOF mass analyzer, thetotal cycle time of the DIA methodology may be reduced relative to theduration of the chromatographic peak, and thus ensuring that the sampleions are suitably sampled. Preferably, the TOF mass analyzer is operatedat a frequency no greater than 1000 Hz, or more preferably 900 Hz, 800Hz or 700 Hz in order to provide sufficient time for the accumulationfragmented ions prior to the injection into the TOF analyzer.Advantageously, by accumulating fragmented ions and mass analyzing thefragmented ions as a packet, the resolution and mass accuracy of theresulting MS2 scans may be increased, while still achieving a suitablefrequency of operation which allows for the analysis of a relativelywide mass range (e.g. 400-1000 m/z) over the duration of achromatographic peak.

Preferably, the present invention utilizes an orbital trapping massanalyzer to perform relatively high resolution MS1 scans of theprecursor ions. Thus, data in the MS1 domain may also be captured withhigh resolution and high mass accuracy. Other mass analyzers of highresolution could be utilized as the first mass analyzer to perform theMS1 scans. For example, an ion cyclotron resonance (ICR) mass analyzeror a time of flight mass analyzer (especially a multi-reflection time offlight mass analyzer).

Preferably, the FTMS mass analyzer performs MS1 scans at a massresolution of at least 50,000, or more preferably at least: 75,000,100,000, 150,000 or 200,000 (resolution at 200 m/z). Where other typesof mass analyzer are used for the MS1 scans of the precursor ions, theyalso preferably perform the MS1 scans at a mass resolution of at least50,000, or more preferably at least: 75,000, 100,000, 150,000 or 200,000(resolution at 200 m/z). By performing MS1 scans at such a massresolution, the MS1 data may be more suitable for analysis using aDDA-type library thus resulting in increased accuracy of precursorquantitation/identification.

Preferably, the present invention utilizes a multiple reflection time offlight mass analyzer (mr-TOF) to perform the plurality of MS2 scans. Byutilizing a mr-TOF, the flight time of the packets of fragmented ionsmay be increased due to the multiple reflections included in the flightpath of the ions packets through the mr-TOF. Utilizing a relatively longflight path, for example in excess of 10 m, or 15 m, or preferably 20 m,or more preferably 25 m is advantageous, as it increases the timeresolution of each packet arriving at the detector in the mr-TOF and mayreduce adverse space-charge effects. Examples of mr-TOF mass analyzersinclude orbital-type mr-TOF analyzers as described in WO 2010/0136533 ortilted-mirror type mr-TOF analyzers as described in WO 2013/110587. Thusthe resolution of MS2 scans using an mr-TOF may be increased withoutrequiring an increase in measurement time for each scan. Accordingly, aplurality of narrow mass window (i.e. less than 5 amu) MS2 scans may beperformed within the duration of a chromatographic peak in accordancewith the methodology of the present invention.

Preferably, the mass range of each segment of precursor ions scanned inthe MS2 domain is not greater than 3 amu, or more preferably not greaterthan: 2.5 amu or 2 amu. By reducing the mass range of each segment ofprecursor ions fragmented for MS2 analysis, the resulting MS2 spectrumhas a reduced number of possible precursor ions from which it wasformed. Accordingly, the complexity of the resulting MS2 spectra may bereduced, thereby improving the subsequent analysis of the MS2 spectrafor quantitation and/or identification of precursor ions.

Preferably, the mass resolution of the TOF mass analyzer is at least40,000 for each of the MS2 scans (resolution at 200 m/z). Morepreferably, the resolution of the TOF analyzer is at least 45,000 or50,000. By providing MS2 scans at such a resolution, the mass accuracyof the MS2 spectra may be matched to the corresponding high resolution,high mass accuracy MS1 scans and/or may be matched to DDA-type databasesfor quantitation and/or identification of the sample.

Preferably, at least X % of the MS2 scans contain more than Y ioncounts, wherein X=30, or 50, or 70, or 90, and Y=200, or 500, or 1000,or 3000, or 5000. By ensuring that at least a specified percentage ofthe MS2 scans have a minimum ion count, the dynamic range of the MS2scans may be increased. Accordingly, the mass accuracy of the resultingMS2 data is increased.

Preferably, the DIA methodology is repeated a number of times over theduration of a chromatographic peak. Accordingly, the cycle time of theDIA measurement may be adapted to be performed a plurality of times overthe duration of a chromatographic peak. By performing the DIAmethodology a number of times over the duration of the chromatographicpeak, the peak may be sampled a number of times, allowing a completepicture of the peak to be established. Preferably, the DIA methodologyis performed at least: 3, 4, 5, 7, 9 or more preferably 10 times overthe duration of a chromatographic peak.

Preferably, the performing of the MS1 scan of the precursor ions acrossthe mass range of interest further comprises separating the precursorions in the mass range of interest into a plurality of mass sub-ranges,and for each mass sub-range of precursor ions mass analyzing thesub-range of precursor ions using the Fourier Transform mass analyzer.As such, the MS1 scan of the precursor ions in the mass range ofinterest is split into a set (plurality) of MS1 scans of a plurality ofmass sub-ranges of the precursor ions, wherein the combination of theplurality of mass sub-ranges make up the mass range of interest. Byseparating the MS1 scan into a set of MS1 scans, space charge effectsassociated with the relatively wide precursor mass range may be reduced.Preferably, the mass range of the precursor ions in each mass sub-rangeis no greater than 300 amu, or more preferably 200 amu, or morepreferably 100 amu. By reducing the mass range of each mass sub-range,space charge effects may be further reduced.

Preferably, the mass range of interest has a mass range of at least 600amu, more preferably at least 800 amu, or more preferably at least 1000amu. For example, the mass range of interest may be 400 m/z to 1000 m/z.By varying the width of the mass range of interest, the time forperforming the DIA analysis may be adapted in order to correspond to theduration of a chromatographic peak. The precursor mass range of interestmay also be chosen based on a desired mass range of precursor ions to beanalyzed.

In a preferred embodiment, the invention comprises: separating theprecursor ions into a plurality of precursor mass sub-ranges across themass range of interest;

wherein for each precursor mass sub-range:

performing an MS1 scan, each MS1 scan comprising:

mass analyzing the precursor ions of the precursor mass sub-range acrossthe mass range of the precursor mass sub-range using a first, preferablyFTMS, mass analyzer; and

ii) performing a plurality of MS2 scans, the plurality of MS2 scanscomprising:

further separating the precursor mass sub-ranges into a plurality ofprecursor mass sub-range segments across the mass range of the precursormass sub-range, each precursor mass sub-range segment having a massrange of no greater than 5 amu;

wherein for each precursor mass sub-range segment:

fragmenting the precursor ions within that precursor mass sub-rangesegment;

performing an MS2 scan of the fragmented ions by:

forming an ion packet from the fragmented ions (preferably byaccumulating the fragmented ions, preferably in an ion trap, to form apacket of fragmented ions); and injecting the ion packet of fragmentedions into a time of flight mass analyzer and mass analyzing thefragmented ions of the fragmented mass sub-range segment.

Preferably, performing an MS2 scan of the set of MS2 scans includescombining fragmented ions from a plurality of precursor mass segmentssuch that the combined fragmented ions are mass analyzed. Preferably, aplurality of precursor mass segments are fragmented together andsubsequently mass analyzed together. Preferably, the data resulting inthis way from the set of MS2 scans is deconvoluted to obtain MS2 spectrafor each of the precursor mass segments. In another preferredembodiment, a plurality of precursor mass segments are fragmentedseparately, e.g. separately in time (sequentially), and subsequentlymass analyzed together (optionally in wherein the set of MS2 scans isdeconvoluted to obtain MS2 spectra for each of the precursor masssegments). By performing MS2 scans of a plurality of precursor masssegments at the same time, the time taken to obtain the MS2 spectra maybe reduced. Additionally, the method allows one or more of the precursormass segments to be mass analyzed as part of more than one combination.Thus, the mass accuracy of the resulting MS2 mass spectra may beimproved as a result of multiple MS2 scans of precursor mass segments.

In accordance with a second aspect of the invention, a mass spectrometeris provided according to claim 14.

In accordance with a third aspect of the invention, a computer programcomprising instructions to cause the mass spectrometer the second aspectof the invention to execute the steps of the method according to thefirst aspect of the invention. For example, the third aspect includes acomputer program comprising instructions to cause the mass spectrometerof any one of claims 14 to 24 to execute the steps of the methodaccording to any one of claims 1 to 13.

In accordance with a fourth aspect of the invention, a computer-readablemedium having stored thereon the computer program of the third aspect isprovided,

A fifth aspect of the invention provides a data independent acquisitionmethod of mass spectrometry for analyzing a sample within a mass rangeof interest, the method being performed within a time period based on awidth of a chromatographic peak of the sample as it elutes from achromatography system. The method comprises a step of ionizing thesample to produce a plurality of precursor ions. The method alsocomprises a step of performing an MS1 scan of the precursor ionsincluding mass analyzing the precursor ions across the mass range ofinterest using a first mass analyzer at a resolution of at least 50,000at m/z=200 for identification and/or quantitation of the precursor ionsin the MS1 domain. The method also comprises a step of performing a setof MS2 scans. Performing the set of MS2 scans comprises segmenting theprecursor ions into a plurality of precursor mass segments, where eachprecursor mass segment has a mass range of no greater than 5 amu. Foreach precursor mass segment, the method comprises fragmenting theprecursor ions within that precursor mass segment and performing an MS2scan of the fragmented ions. Performing the MS2 scan of the fragmentedions comprises forming an ion packet from the fragmented ions andinjecting the ion packet of fragmented ions into a time of flight massanalyzer and mass analyzing the fragmented ions. The time of flight massanalyzer performs each of the MS2 scans at a resolution of at least40,000 at m/z=200. Preferably the time of flight mass analyzer performsthe MS2 scans at a frequency of at least 100 Hz. For example, the firstmass analyzer according to the fifth aspect may be a Fourier Transformmass analyzer or a Time of Flight mass analyzer which is distinct fromthe Time of flight mass analyzer used to perform the MS2 scans.

The advantages and optional features of the first aspect of theinvention as discussed above applies equally to the second, third,fourth, and fifth aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be put into practice in a number of ways and specificembodiments will now be described by way of example only and withreference to the Figures in which:

FIG. 1 shows a schematic diagram of a mass spectrometer suitable forcarrying out methods in accordance with embodiments of the invention;

FIG. 2 shows a diagram representative of the MS1 and MS2 scans carriedin accordance with an embodiment of the invention;

FIG. 3 shows an exemplary flow chart of the DIA methodology according toan embodiment of the present invention;

FIG. 4 shows a schematic diagram of an alternative mass spectrometersuitable for carrying out method in accordance with embodiments of theinvention;

FIG. 5a shows an example of the DIA methodology according to anembodiment of the present invention superimposed onto thecharacteristics of a chromatographic peak;

FIG. 5b shows a schematic diagram of timings associated with the loadingof the mass analyzers in accordance with embodiments of the invention;

FIG. 6 shows a schematic diagram of a further alternative massspectrometer suitable for carrying out method in accordance withembodiments of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Herein the term mass may be used to refer to the mass-to-charge ratio,m/z. The resolution of a mass analyzer is to be understood to refer tothe resolution of the mass analyzer as determined at a mass to chargeratio of 200 unless otherwise stated.

FIG. 1 shows a schematic arrangement of a mass spectrometer 10 suitablefor carrying out methods in accordance with embodiments of the presentinvention.

In FIG. 1, a sample to be analyzed is supplied (for example from anautosampler) to a chromatographic apparatus such as a liquidchromatography (LC) column (not shown in FIG. 1). One such example of anLC column is the Thermo Fisher Scientific, Inc ProSwift monolithiccolumn which offers high performance liquid chromatography (HPLC)through the forcing of the sample carried in a mobile phase under highpressure through a stationary phase of irregularly or spherically shapedparticles constituting the stationary phase. In the HPLC column, samplemolecules elute at different rates according to their degree ofinteraction with the stationary phase.

A chromatograph may be produced by measuring over time the quantity ofsample molecules which elute from the HPLC column using a detector (forexample a mass spectrometer). Sample molecules which elute from the HPLCcolumn will be detected as a peak above a baseline measurement on thechromatograph. Where different sample molecules have different elutionrates, a plurality of peaks on the chromatograph may be detected.Preferably, individual sample peaks are separated in time from otherpeaks in the chromatogram such that different sample molecules do notinterfere with each other.

On a chromatograph, a presence of a chromatographic peak corresponds toa time period over which the sample molecules are present at thedetector. As such, a width of a chromatographic peak is equivalent to atime period over which the sample molecules are present at a detector.Preferably, a chromatographic peak has a Gaussian shaped profile, or canbe assumed to have a Gaussian shaped profile. Accordingly, a width ofthe chromatographic peak can be determined based on a number of standarddeviations calculated from the peak. For example, a peak width may becalculated based on 4 standard deviations of a chromatographic peak.Alternatively, a peak width may be calculated based on the width at halfthe maximum height of the peak. Other methods for determining the peakwidth known in the art may also be suitable.

The sample molecules thus separated via liquid chromatography are thenionized using an electrospray ionization source (ESI source) 20 which isat atmospheric pressure.

Sample ions then enter a vacuum chamber of the mass spectrometer 10 andare directed by a capillary 25 into an RF-only S lens 30. The ions arefocused by the S lens 30 into an injection flatapole 40 which injectsthe ions into a bent flatapole 50 with an axial field. The bentflatapole 50 guides (charged) ions along a curved path through it whileunwanted neutral molecules such as entrained solvent molecules are notguided along the curved path and are lost.

An ion gate (TK lens) 60 is located at the distal end of the bentflatapole 50 and controls the passage of the ions from the bentflatapole 50 into a downstream mass selector in the form of a quadrupolemass filter 70. The quadrupole mass filter 70 is typically but notnecessarily segmented and serves as a band pass filter, allowing passageof a selected mass number or limited mass range while excluding ions ofother mass to charge ratios (m/z). The mass filter can also be operatedin an RF-only mode in which it is not mass selective, i.e. it transmitssubstantially all m/z ions. For example, the quadrupole mass filter 70may be controlled by the controller 130 to select a range of mass tocharge ratios to pass of the precursor ions which are allowed to mass,while the other ions in the precursor ion stream are filtered.Alternatively, the S lens 30 may be operated as an ion gate and the iongate (TK lens) 60 may be a static lens.

Although a quadrupole mass filter is shown in FIG. 1, the skilled personwill appreciate that other types of mass selection devices may also besuitable for selecting precursor ions within the mass range of interest.For example, an ion separator as described in US-A-2015287585, an iontrap as described in WO-A-2013076307, an ion mobility separator asdescribed in US-A-2012256083, an ion gate mass selection device asdescribed in WO-A-2012175517, or a charged particle trap as described inUS799223, the contents of which are hereby incorporated by reference intheir entirety. The skilled person will appreciate that other methodsselecting precursor ions according to ion mobility, differentialmobility and/or transverse modulation may also be suitable.

The isolation of a plurality of ions of different masses or mass rangesmay also be performed using the method known as synchronous precursorscanning (SPS) in an ion trap. Furthermore, in some embodiments, morethan one ion selection or mass selection device may be provided. Forexample, a further mass selection device may be provided downstream ofthe fragmentation chamber 120. In this way, MS³ or MS^(n) scans can beperformed if desired (typically using the TOF mass analyzer for massanalysis).

Ions then pass through a quadrupole exit lens/split lens arrangement 80and into a first transfer multipole 90. The first transfer multipole 90guides the mass filtered ions from the quadrupole mass filter 70 into acurved linear ion trap (C-trap) 100. The C-trap (first ion trap) 100 haslongitudinally extending, curved electrodes which are supplied with RFvoltages and end caps that to which DC voltages are supplied. The resultis a potential well that extends along the curved longitudinal axis ofthe C-trap 100. In a first mode of operation, the DC end cap voltagesare set on the C-trap so that ions arriving from the first transfermultipole 90 are captured in the potential well of the C-trap 100, wherethey are cooled. The injection time (IT) of the ions into the C-trapdetermines the number of ions (ion population) that is subsequentlyejected from the C-trap into the mass analyzer.

Cooled ions reside in a cloud towards the bottom of the potential welland are then ejected orthogonally from the C-trap towards the first massanalyzer 110. As shown in FIG. 1, the first mass analyzer is an orbitaltrapping mass analyzer 110, for example the Orbitrap® mass analyzer soldby Thermo Fisher Scientific, Inc. The orbital trapping mass analyzer 110has an off center injection aperture and the ions are injected into theorbital trapping mass analyzer 110 as coherent packets, through the offcenter injection aperture. Ions are then trapped within the orbitaltrapping mass analyzer by a hyperlogarithmic electric field, and undergoback and forth motion in a longitudinal direction while orbiting aroundthe inner electrode.

The axial (z) component of the movement of the ion packets in theorbital trapping mass analyzer is (more or less) defined as simpleharmonic motion, with the angular frequency in the z direction beingrelated to the square root of the mass to charge ratio of a given ionspecies. Thus, over time, ions separate in accordance with their mass tocharge ratio.

Ions in the orbital trapping mass analyzer are detected by use of animage detector (not shown) which produces a “transient” in the timedomain containing information on all of the ion species as they pass theimage detector. The transient is then subjected to a Fast FourierTransform (FFT) resulting in a series of peaks in the frequency domain.From these peaks, a mass spectrum, representing abundance/ion intensityversus m/z, can be produced.

In the configuration described above, the sample ions (morespecifically, a mass range segment of the sample ions within a massrange of interest, selected by the quadrupole mass filter) are analyzedby the orbital trapping mass analyzer without fragmentation. Theresulting mass spectrum is denoted MS1.

Although an orbital trapping mass analyzer 110 is shown in FIG. 1, otherFourier Transform mass analyzers may be employed instead. For example aFourier Transform Ion Cyclotron Resonance (FTICR) mass analyzer may beutilized as mass analyzer for the MS1 scans. Mass analyzers, such as theorbital trapping mass analyzer and Ion Cyclotron Resonance massanalyzer, may also be used in the invention even where other types ofsignal processing than Fourier transformation are used to obtain massspectral information from the transient signal (see for example WO2013/171313, Thermo Fisher Scientific).

In a second mode of operation of the C-trap 100, ions passing throughthe quadrupole exit lens/split lens arrangement 80 and first transfermultipole 90 into the C-trap 100 may also continue their path throughthe C-trap and into the fragmentation chamber 120. As such, the C-trapeffectively operates as an ion guide in the second mode of operation.Alternatively, cooled ions in the C-trap 100 may be ejected from theC-trap in an axial direction into the fragmentation chamber 120. Thefragmentation chamber 120 is, in the mass spectrometer 10 of FIG. 1, ahigher energy collisional dissociation (HCD) device to which a collisiongas is supplied. Precursor ions arriving into the fragmentation chamber120 collide with collision gas molecules resulting in fragmentation ofthe precursor ions into fragment ions.

Although an HCD fragmentation chamber 120 is shown in FIG. 1, otherfragmentation devices may be employed instead, employing such methods ascollision induced dissociation (CID), electron capture dissociation(ECD), electron transfer dissociation (ETD), photodissociation, and soforth.

Fragmented ions may be ejected from the fragmentation chamber 120 at theopposing axial end to the C-trap 100. The ejected fragmented ions passinto a second transfer multipole 130. The second transfer multipole 130guides the fragmented ions from the fragmentation chamber 120 into anextraction trap (second ion trap) 140. The extraction trap 140 is aradio frequency voltage controlled trap containing a buffer gas. Forexample, a suitable buffer gas is argon at a pressure in the range5×10⁻⁴ mBar to 1×10⁻² mBar. The extraction trap has the ability toquickly switch off the applied RF voltage and apply a DC voltage toextract the trapped ions. A suitable flat plate extraction trap, alsoreferred to as a rectilinear ion trap, is further described in U.S. Pat.No. 9,548,195 (B2). Alternatively, a C-trap may also be suitable for useas a second ion trap.

The extraction trap 140 is provided to form an ion packet of fragmentedions, prior to injection into the time of flight mass analyzer 150. Theextraction trap 140 accumulates fragmented ions prior to injection ofthe fragmented ions into the time of flight mass analyzer 150.

Although an extraction trap (ion trap) is shown in the embodiment ofFIG. 1, the skilled person will appreciate that other methods of formingan ion packet of fragmented ions will be equally suitable for thepresent invention. For example, relatively slow transfer of ions througha multipole can be used to affect bunching of ions, which cansubsequently be ejected as a single packet to the TOF mass analyzer.Alternatively orthogonal displacement of ions may be used to form apacket. Further details of these alternatives are found in US20030001088A1 which describes a travelling wave ion bunching method, the contentsof which are herein incorporated by reference.

In FIG. 1, the time of flight mass analyzer 150 shown is a multiplereflection time of flight mass analyzer (mr-TOF) 150. The mr-TOF 150 isconstructed around two opposing ion mirrors 160, 162, elongated in adrift direction. The mirrors are opposed in a direction that isorthogonal to the drift direction. The extraction trap 140 injects ionsinto the first mirror 160 and the ions then oscillate between the twomirrors 160, 162. The angle of ejection of ions from the extraction trap140 and additional deflectors 170, 172 allow control of the energy ofthe ions in the drift direction, such that ions are directed down thelength of the mirrors 160, 162 as they oscillate, producing a zig-zagtrajectory. The mirrors 160, 162 themselves are tilted relative to oneanother, producing a potential gradient that retards the ions' driftvelocity and causes them to be reflected back in the drift dimension andfocused onto a detector 180. The tilting of the opposing mirrors wouldnormally have the negative side-effect of changing the time period ofion oscillations as they travel down the drift dimension. This iscorrected with a stripe electrode 190 (to act as a compensationelectrode) that alters the flight potential for a portion of theinter-mirror space, varying down the length of the opposing mirrors 160,162. The combination of the varying width of the stripe electrode 190and variation of the distance between the mirrors 160, 162 allows thereflection and spatial focusing of ions onto the detector 180 as well asmaintaining a good time focus. A suitable mr-TOF 150 for use in thepresent invention is further described in US 2015028197 (A1), thecontents of which are hereby incorporated by reference in its entirety.

Ions accumulated in the extraction trap are injected into the mr-TOF 150as a packet of ions, once a predetermined number of ions have beenaccumulated in the extraction trap. By ensuring that each packet of ionsinjected into the mr-TOF 150 has at least a predetermined (minimum)number of ions, the resulting packet of ions arriving at the detectorwill be representative of the entire mass range of interest of the MS2spectrum. Accordingly, a single packet of fragmented ions is sufficientto acquire MS2 spectra of the fragmented ions. This represent anincreased sensitivity compared to conventional acquisition of time offlight spectra in which multiple spectra typically are acquired andsummed for each given mass range segment. Preferably, a minimum totalion current (TIC) in each narrow mass window is accumulated in theextraction trap before ejection to the time of flight mass analyzer.Preferably, at least N spectra (scans) are acquired per second in theMS2 domain by the time of flight mass analyzer, wherein N=50, or morepreferably 100, or 200, or more.

Preferably, at least X % of the MS2 scans contain more than Y ion counts(wherein X=30, or 50, or 70, or most preferably 90, or more, and Y=200,or 500, or 1000, or 2000, or 3000, or 5000, or more). Most preferably,at least 90% of the MS2 scans contain more than 500 ion counts, or morepreferably more than 1000 ion counts. This provides for an increaseddynamic range of MS2 spectra. The desired ion counts for each of the MS2scans may be provided by adjusting the number ions included in eachpacket of fragmented ions. For example, in the embodiment of FIG. 1, theaccumulation time of the extraction trap may be adjusted to ensure thata sufficient number of ions have been accumulated. As such, thecontroller 195 may be configured to determine that a suitable packet offragmented ions has been formed when either a predetermined number ofions are present in the extraction trap, or a predetermined period oftime has passed. The predetermined period of time may be specified inorder to ensure that the time of flight mass analyzer operates at thedesired frequency when the flow of ions to the extraction trap isrelatively low.

The mass spectrometer 10 is under the control of a controller 195 which,for example, is configured to control the timing of ejection of thetrapping components, to set the appropriate potentials on the electrodesof the quadrupole etc so as to focus and filter the ions, to capture themass spectral data from the orbital trapping device 110, to capture themass spectral data from the mr-TOF 150, control the sequence of MS1 andMS2 scans and so forth. It will be appreciated that the controller 195may comprise a computer that may be operated according to a computerprogram comprising instructions to cause the mass spectrometer toexecute the steps of the method according to the present invention.

It is to be understood that the specific arrangement of components shownin FIG. 1 is not essential to the methods subsequently described. Indeedother arrangements for carrying out the DIA methods of embodiments ofthe present invention are suitable.

An exemplary embodiment of the method will now be described withreference to FIGS. 2 and 3, in which sample molecules are supplied froma liquid chromatography (LC) column as part of the exemplary apparatusdescribed above (as shown in FIG. 1).

In the exemplary embodiment of the invention, the sample ions aresupplied from the LC column such that the DIA methodology according tothe present invention acquires data about the sample over a durationcorresponding to a duration of a chromatographic peak of the samplesupplied from the LC column. As such, the controller 195 is configuredto perform the method within a time period corresponding to the width(duration) of a chromatographic peak at its base.

As shown in FIG. 2, an orbital trapping mass analyzer (denoted“Orbitrap”) is utilized to perform a plurality of MS1 scans across amass range of interest. For example, as shown in FIG. 2, the mass rangeof interest to be analyzed is 400-1000 m/z. Within the mass range ofinterest, a plurality of MS1 scans are performed using mass sub rangesof precursor ions of the mass range of interest. Alternatively, a singleMS1 scan may be performed using precursor ions from the entire massrange of interest (i.e. 400-1000 amu in this example).

In order to perform a single MS1 scan, sample molecules from an LCcolumn are ionized using the ESI source 20. Sample ions subsequentlyenter the vacuum chamber of the mass spectrometer 10. The sample ionsare directed by through capillary 25, RF-only lens 30, injectionflatapole 40, bent flatapole 50 and into the quadrupole mass filter 70in the manner as described above. The quadrupole mass filter 70 iscontrolled by the controller 195 to filter the sample ions according tothe selected precursor mass sub-range of interest. For example, as shownin FIG. 2 MS1 scans are performed across a mass range of interest from400 m/z to 1000 m/z in precursor mass sub-ranges of 400-500 m/z, 500-600m/z . . . to 900-1000 m/z.

Ions then pass through the quadrupole exit lens/split lens arrangement80, through the transfer multipole 90 and into the C-trap 100 where theyare accumulated. From the C-trap 100, (precursor) sample ions of themass range segment may be injected in to the orbital trapping massanalyzer 110. Once ions are stabilized inside the orbital trapping massanalyzer, the MS1 scan is performed by using the image current detectorto detect the ions present in the orbital trapping mass analyzer 110.The detection of the ions in the orbital trapping mass analyzer isconfigured to be performed with a relatively high resolution for the MS1scan (relative to the resolution of the MS2 scans). For example, aresolution (R) of at least 50,000, or preferably at least 100,000 may beused for each MS1 scan (see Resolution R=120,000 in FIG. 2).

By using a Fourier Transform mass analyzer (for example an orbitaltrapping mass analyzer), the MS1 scans are performed with a high degreeof mass accuracy. Preferably, the MS1 scans are performed with a massaccuracy of less than 5, or more preferably 3 parts per million (ppm).Parts per million mass accuracy Δm of a mass analyzer may be determinedas the difference between the measured mass of an ion m, and the actualmass of an ion m_(a), divided by the actual mass of the ion, multipliedby 10⁶, as shown below:

${\Delta \; m} = {\frac{( {m_{i} - m_{a}} )}{m_{a}} \times 10^{6}}$

In tandem with the MS1 scans, a plurality of MS2 scans are performed, asshown in FIG. 2. The MS2 scans are performed using a time of flight massanalyzer, or more preferably a mr-TOF, as shown in FIG. 2.

In order to perform a single MS2 scan of a mass range segment, samplemolecules from an LC column are ionized and injected into the massspectrometer in a similar manner to the MS1 scan. The sample ions forthe MS2 scan progress through the capillary 25, RF-only lens 30,injection flatapole 40, bent flatapole 50 and into the quadrupole massfilter 70 in a similar manner to the sample ions for the MS1 scan.

Once the sample ions for the MS2 scan reach the quadrupole mass filter70, the quadrupole mass filter 70 is controlled by the controller 195 tofilter the sample ions according to the relatively narrow mass rangesegment being scanned. Each precursor mass range segment has a massrange of no greater than 5 amu, or preferably no greater than 3 amu, ormore preferably no greater than 2 amu (as shown in FIG. 2).

The (filtered mass range segment) precursor ions pass from thequadrupole mass filter 70 through to the C-trap 100 as described abovefor the MS1 scan. The controller 195 then controls the C-trap to allowthe precursor ions to pass through in an axial direction towards thefragmentation chamber 120.

In the HCD fragmentation chamber 120, the precursor ions collide withcollision gas molecules which results in the fragmentation of theprecursor ions into fragment ions.

The fragmented ions for the mass range segment are then ejected from thefragmentation chamber at the opposing axial end to the C-trap 100. Theejected fragmented ions pass into a second transfer multipole 130. Thesecond transfer multipole 130 guides the fragmented ions from thefragmentation chamber 120 into an extraction trap (second ion trap) 140where they are accumulated. The fragmented ions may be accumulated inthe extraction trap 140 for a predetermined time.

Fragmented ions are then injected from the extraction trap injected intothe mr-TOF. The prior accumulation of fragmented ions in the extractiontrap allows the fragmented ions to be injected as a packet into themr-TOF. The packet of ions travels along the flight path of the mr-TOFundergoing multiple reflections before being detected at the detector.The varying arrival times of the fragmented ions within the packetallows a MS2 mass spectrum for the packet of fragmented ions to begenerated. The length of the flight path of the mr-TOF in combinationwith the time resolution of the detector allows the mr-TOF to performMS2 scans at a resolution in excess of 40,000 (see R=50,000 in FIG. 2).

One benefit of the packet-based approach to the analysis of thefragmented ions in the MS2 domain, is that once the accumulated ions areejected from the extraction trap, the fragmented ions for the next massrange segment can begin to fill the extraction trap. As such, fragmentedions from one mass range segment can be travelling through the mr-TOFwhile the next mass range segment of fragmented ions is beingaccumulated. Thus, a greater number of ion counts in each MS2 scan canbe achieved within a chromatographic peak due to the use of theaccumulated packet based injection of fragmented ions into the mr-TOFfrom the extraction trap.

According to the exemplary embodiment, in a single cycle of the DIAmethodology, the controller 195 controls the mass spectrometer 10 toperform the plurality of MS1 scans of the mass sub-ranges for the massrange of interest, and in tandem the plurality of MS2 scans of the massrange segments over the mass range of interest. In order to acquire amore accurate sample of the chromatographic peak, the controller 195 mayrepeat the cycle of the DIA methodology a number of times over theduration of the chromatographic peak. For example, a single cycle of theDIA methodology may take around 1.5 s to perform. As such, the cycle maybe performed at least 7 times, or more preferably at least 9 times overthe duration of a chromatographic peak. This enables the MS1 and/or MS2spectral data to be used for quantitation of the eluting sample in thechromatographic peak.

A further exemplary embodiment of the method of the present invention isdescribed in the flow chart of FIG. 3. As shown in FIG. 3, a pluralityof MS1 and MS2 scans may be performed in tandem. In the furtherexemplary embodiment, sample molecules may be supplied from a liquidchromatography (LC) column, for example as part of the exemplaryapparatus described above (as shown in FIG. 1).

According to the further exemplary embodiment, at a time T1, precursorions travel from the ESI 20 source through to the C-trap 100, forexample in a manner as described previously. Along the way, theprecursor ions are filtered by the quadrupole mass selector 70 to leaveions in a first mass range segment (e.g. 400-500 m/z).

Once the C-trap 100 is filled, at a time T2, precursor ions in the firstmass range segment are ejected to the orbital trapping mass analyzer 110for performing the MS1 scan. The MS1 scan in the orbital trapping massanalyzer 110 is performed in tandem (parallel) with the plurality of MS2scans in the mr-TOF 150. As shown, one MS1 scan in the orbital trappingmass analyzer for one of the mass sub-ranges (at resolution of 120,000)takes 0.25 seconds.

At a time T3, a packet of precursor ions is passed from the ESI source20, filtered through the quadrupole mass selector through the C-Trap 100and to the collision cell. The quadrupole mass selector 70 filters theions to define a first mass range segment (e.g. 400-402 m/z as shown inFIG. 3). Effectively, the C-Trap 100 operates as an ion guide except attimes T1 and T2.

At a time T4, the first mass range segment of precursor ions are thensent to the collision cell 120, where they are fragmented. Thefragmented ions are then sent onwards to the mr-TOF 150 where they areaccumulated in the extraction trap 140

At a time T5, the extraction trap 140 ejects a packet of fragmented ionsfor travel along the flight path.

At a time T6, the mr-TOF 150 detects the fragmented ions at the detector180. As shown, one MS2 scan in the time of flight mass analyzer for oneof the mass range segments (at resolution of 50,000, 500 Hz) takes 2milliseconds. According to the embodiment of FIG. 3, although the MS2scan takes 2 ms to perform, the time taken to fragment the ion (3 ms),and accumulate the ions in the extraction trap and eject them (2 ms)takes a total time period of 5 ms. Accordingly the time period forperforming a single MS2 scan according to this embodiment is set by this5 ms time period. In other embodiments, the time for performing thesemethod steps may be varied in order to vary the frequency of performingthe MS2 scans.

At a time T7, a subsequent set of precursor ions is filtered to usingthe next narrow mass window for the next MS2 scan (i.e. mass range402-404 m/z), fragmented in the collision cell (time T8), ejected fromthe extraction trap (time T9) and detected at the detector (time T10).As such, the DIA apparatus processes multiple packets of ions for theMS2 scans at once at different points in the system. For example, theextraction trap is being filled with ions while the previous ion packetis flying through the mr-TOF 150. As such, the parallel TOF MS2acquisition step is performed concurrently with the steps of fragmentingthe ions and accumulating the ions for ejection into the TOF. In someembodiments, it is preferable that while fragmented ions are cooling inthe extraction trap 140 a further ion packet should be fragmenting inthe collision cell 120.

Thus, as shown in FIG. 3, MS2 scans are performed for each narrow masswindow across the precursor mass sub-range (e.g. 400-500 m/z).

Once the MS1 scan and the plurality of MS2 scans for the mass sub-range400-500 m/z are complete, the MS1 scan and MS2 scans above are repeatedfor the next mass sub-range (e.g. 500-600 m/z). Accordingly, in theexemplary embodiment of FIG. 3, performing the MS1 scan and plurality ofMS2 scans for each mass sub-range takes 250 ms. Thus, the complete DIAscan of the mass range of interest may be completed in a cycle time of1.5 s. By performing a high resolution, high mass accuracy DIAmethodology with a cycle time of 1.5 s, the DIA methodology of thepresent invention may be performed a number of times over the durationof a chromatographic peak. For example, the method of the presentinvention may be repeated at least: 3, 5, 7, 9, or more preferably atleast 10 times across the duration of the chromatographic peak. Thus, achromatographic peak may be fully sampled using a DIA methodologyaccording to the present invention.

The precursor ion spectra acquired at high resolution and high massaccuracy in the orbital trapping mass analyzer can be used to produce aprecursor ion candidate list using a standard non-fragment ion-database.In-silico fragmentation of the candidates in the list produces withinthe narrow mass range of interest a library against which fragmentationspectra are compared and/or quantified as known in the art in order toprovide identification of the precursor ions.

The skilled person will understand from FIG. 3, that the DIA methodologyof the present invention may be applied to any mass range of interest.For example, a mass range of interest may include a mass to charge ratioof at least: 50, 100, 200, 300, 400 or 500 m/z in order to analyzeprecursor ions of a relatively small m/z. A mass range of interest mayalso include a mass to charge ratio of up to: 800, 900, 1000, 1200,1400, 1600, 2000 or 2500 m/z in order to analyze precursor ions of arelatively large m/z.

In one alternative embodiment, a set of MS2 scans may be acquired bymultiplexing precursor ions from a plurality of different mass rangesegments in a single MS2 analysis. In this alternative method ofperforming a set of MS2 scans, the MS1 scans may be acquired accordingto any of the methodologies described previously.

In the alternative embodiment, for each MS2 scan making up the set ofMS2 scans, a plurality of mass range segments are selected. A differentcombination of mass range segments is selected for each scan in the setof MS2 scans, and also ensures that every mass range segment is selectedat least once. In the alternative embodiment, precursor ions from theselected combination of mass range segments may be fragmented andanalyzed together as part of a single MS2 scan. The precursor ions ineach mass range segment may be fragmented separately and the resultingfragmented ions formed (accumulated) into a packet together to bescanned together in the TOF mass analyzer. Alternatively, the precursorions in the plurality of mass range segments to be combined may befragmented together, and then scanned together in the TOF mass analyzer.For example, a first MS2 scan may be performed on precursor ions frommass range segments with mass to charge ratios of 400-402, 420-422 and440-442. A second MS2 scan may then be performed on a differentcombination of mass range segments. Once all the MS2 scans in the sethave been performed, the MS2 data may be deconvoluted in order obtaindata suitable for identification and/or quantitation of the sample.Further details of the methodology and the deconvolution process may befound in “Multiplexed MS/MS for improved data-independent acquisition”,Egertson J D et al, Nature Methods 10, 29 Aug. 2012,doi:10.1038mmeth.2528, the contents of which is herein incorporated byreference.

In a further alternative embodiment, the mass spectrometer according tothe present invention may be provided in a branched path arrangement,for example as shown in the embodiment in FIG. 4. In the embodiment ofFIG. 4, an ion source 200 is coupled to a mass selection device 210.Such an arrangement may be provided by the ESI ion source 20 and itsrespective couplings to the quadrupole mass filter 70 as shown in theembodiment of FIG. 1 for example.

As shown in FIG. 4, the output of the mass selection device 210 iscoupled to the branched ion path 220. The branched ion path directs ionsoutput from the mass selection device along one of two paths. A firstpath 222 directs ions to a C-trap 230 where ions are collected foranalysis by a Fourier Transform mass analyzer, for example an orbitaltrapping mass analyzer 240 in the MS1 domain. A second path 224 directsions to a fragmentation chamber 250 for fragmentation of ions andsubsequent mass analysis in the MS2 domain. The branched ion path mayuse an rf voltage to direction ions down either the first path 222 orthe second path 224. The branched ion path may be a branched RFmultipole. A branched ion path suitable for use in the embodiment ofFIG. 4 is further described in U.S. Pat. No. 7,420,161.

According to the alternative embodiment in FIG. 4, the branched ion pathmay be used to direct ions to a C-trap 230 for MS1 analysis or to afragmentation chamber 250 for MS2 analysis. Fragmented ions ejected fromthe fragmentation chamber 250 may be accumulated in ion extraction trap260, before being injected into mr-TOF analyzer 270 as a packet. Assuch, the arrangement of the fragmentation chamber 250, ion trap 260 andmr-TOF 270 may be provided by a similar arrangement as described in FIG.1.

Thus, according to the alternative embodiment in FIG. 4, ions may bedirected for MS2 analysis without requiring the C-trap 230 supplying theMS1 orbital trapping mass analyzer 240 to be empty. Such a configurationmay allow increased parallelisation of the MS1 and MS2 scans. As such, agreater proportion of the duration of a chromatographic peak may beavailable for carrying out MS2 scans. Furthermore, in thisconfiguration, a number of loadings or fills can be accumulated in theC-trap 230 before analysis in the orbital trapping mass analyzer 240, asshown in FIGS. 5a and 5b , which are discussed further below. In suchembodiments, the loading (filling) of the C-trap 230 can be split intoseveral small fills while the orbital trap mass analyzer 240 isscanning, thereby to obtain a population of ions that is morerepresentative of the ions from across the entire peak.

FIGS. 5a and 5b show schematic diagrams of a method of DIA analysisusing the branched path configuration of the mass analyzers. FIG. 5ashows a representation of a chromatographic peak of a sample as itelutes from a chromatographic apparatus. The dots superimposed on thepeak represent time periods in which an MS1 scan is started, while thelines are representative time periods in which individual MS2 scans areperformed. As such, the dots correspond to starting points for separatecycles of the DIA methodology.

By utilizing the branched path arrangement, the C-trap 230 is no longerin the path of the supply of ions to the fragmentation chamber forperforming the MS2 scans. Accordingly, the C-trap 230 according to thebranched path embodiment may be loaded over an extended time periodusing a plurality of smaller filling steps.

FIG. 5b shows a schematic representation of the operation of the C-trapand Orbitrap over time for performing MS1 scans according to theembodiment of FIG. 5. The axis denoted “orbi LT ”represents the ion flowinto the C-trap 230 over time, while the axis denoted “orbitrap”represents the operation of the orbital trapping mass analyzer forperforming an MS1 scan over time. As shown in FIG. 5b , ions flow intothe C-trap 230 at a number of relatively short periods over the durationof an MS1 scan by the orbital trapping mass analyzer. As such, theC-trap 230 is filled using a plurality of fills distributed over aperiod of time corresponding to the duration of an MS1 scan. As such, ina sequence of MS1 scans, the ions for the following MS1 scan are loadedinto the C-trap 230 while the current MS1 scan is performed.Advantageously, by loading the C-trap 230 in this manner, the ions inthe C-trap 240 may be more representative of the sample over theduration of loading.

As further shown in FIG. 5b , the loading of the C-trap 230 is performedin parallel with the performance of the loading of the extraction trapfor the MS2 scans. The axis in FIG. 5b denoted “TOF LT” represents theion flow into the extraction trap 260 over time. The axis denoted “TOF”in FIG. 5b represents the operation of the TOF mass analyzer performingMS2 scans over time. As shown in FIG. 5b , each filling of theextraction trap has a corresponding MS2 scan. The skilled person willappreciate that the graph is not to scale and is not intended toaccurately represent the relative frequencies of the performance of theMS1 and MS2 scans.

A further alternative embodiment of the invention is disclosed in FIG.6. FIG. 6 depicts a schematic diagram of a tandem mass spectrometer 300including an orbital trapping mass analyzer 310 and a time of flightmass analyzer 320 in a branched path configuration.

The embodiment in FIG. 6 includes an ion source 330 and ion guide 340which supply precursor ions to a mass selector 350. Such an arrangementmay be provided by the ESI ion source 20 and its respective couplings tothe quadrupole mass filter 70 as shown in the embodiment of FIG. 1 forexample.

As shown in FIG. 6, a branched ion path 360 guides ions from the massselector 350 to a C-trap 370 and/or an extraction trap 380. The C-trap370 supplies ion to the orbital trapping mass analyzer 310 for MS1scans, while the extraction trap 380 supplies ions to the time of flightmass analyzer 320. For example, a similar arrangement to the embodimentdisclosed in FIG. 4 may be provided.

FIG. 6 additionally includes a dual linear trap 400,410. The dual lineartrap is connected downstream of the C-trap 370 between the C-trap 370and the extraction trap 380 for the time of flight mass analyzer. Thedual linear trap may be connected to the C-trap 370 and the extractiontrap 380 by ion guides 420, 430. Ion guide 430 may be a branched ionpath which merges with the ion path from the mass selector 350 in orderto connect to the extraction trap 380.

The dual linear trap 400, 410 may be provided for fragmentation and/ormass isolation of the ions. For example, a first ion trap 400 may beprovided as a high energy collision dissociation chamber. A second iontrap, downstream of the first ion trap may be provided as a lowcollision dissociation chamber. By including a second dissociationchamber, fragmented ions may readily be fragmented again in the secondchamber in order to perform MS3 analysis. Ions may be repeatedlyisolated and fragmented for MS^(n) analysis. The dual linear trap mayalso allow for fragmentation by collision induced dissociation (CID),electron capture dissociation (ECD), electron transfer dissociation(ETD), ultraviolent photo dissociation (UVPD), and so forth. Furtherdetails of a suitable dual ion trap may be found in U.S. Pat. No.8,198,580, the contents of which is herein incorporated by reference inits entirety.

Advantageously, by providing a branched path directly to the extractiontrap 380 from the mass selector 350, ions may be more efficientlytransferred from the mass selector 350 to the extraction trap.

The above described mass spectrometers may also be well suited tocarrying out the DIA methodologies described in GB 1701857.3 (thecontents of which is hereby incorporated by reference in its entirety),wherein the MS2 scans are performed using the time of flight analyzer ofthe above described mass spectrometers, and MS1 scans are performedusing the above described orbital trapping mass analyzers.

It will be appreciated that other embodiments of ion injection into thetime of flight mass analyzer may be used in place of the extractiontrap, although they may not be as advantageous. For example, anorthogonal accelerator may be used to inject packets of ions into thetime of flight mass analyzer. However, the ion counts in each MS2spectrum, and thus dynamic range and sensitivity, in such embodimentswill typically be lower than can be obtained using the extraction trapto accumulate ions before each injection into the time of flight massanalyzer. In a further alternative embodiment, a slow transfer multipolemay be used in place of the extraction trap to inject packets of ionsinto the time of flight mass analyzer. In this way, ions may be bunchedin the multipole (and not lost) before each packet is injected into theTOF analyzer.

Advantageously, the present invention may be used to create a highresolution, high mass accuracy DIA workflow, which can deliver highconfidence of identification, and better precision of quantitation thanapproaches previously known in the art. In some embodiments, the presentinvention is capable of delivering 100% identification of all detectablefeatures in a sample in the MS1 level and with a higher degree ofsensitivity than quantitation in the MS2 level, as quantitation isperformed prior to fragmentation of the sample precursors (i.e. withunfragmented precursors). For example, quantitation of precursor ionsmay be performed in the MS1 domain using a “library free” approach, thusreducing the requirements on post-processing of the acquired data. Onemethod for analyzing DIA MS1 scan data and quantitating precursor ionsusing a library free approach is described in “DIA-Umpire: comprehensivecomputational framework for data independent acquisition proteomics”,Tsou et al, Nat Methods, March 2015 p258-264.

For successful identification of precursor ion information by themethodology of the present invention, the MS1 scans are performed with aresolution of preferably at least 50,000, or more preferably at least100,000K, or better still at least 200,000 and preferably 1-2 ppm orbetter in mass accuracy, while in the ion fragment (MS2) spectra atleast 40,000 or more preferably at least 50,000 resolution andpreferably 5-10 ppm or better mass accuracy is preferable.

Furthermore, the relatively narrow mass windows of each of the MS2 scans(<5 amu), together with their relatively high mass resolution and massaccuracy, allows the DIA methodology of the present invention to beutilized in combination with the traditional database search approachknown from data dependent analysis (DDA). As such, DDA algorithms, orother spectral library-free algorithms may be utilized to perform theDIA data analysis and provide high confidence identification ofprecursors in the sample. In this way, the DIA methodology of thepresent invention does not require the building up a spectral library inadvance, as is currently the case in known DIA methodologies.

1. A data independent acquisition method of mass spectrometry foranalyzing a sample within a mass range of interest, the method beingperformed within a time period based on a width of a chromatographicpeak of the sample as it elutes from a chromatography system andcomprising the steps of: ionizing the sample to produce a plurality ofprecursor ions; selecting precursor ions within a mass range of interestto be analyzed; performing at least one MS1 scan of the precursor ionscomprising: mass analyzing precursor ions across the mass range ofinterest using a Fourier Transform mass analyzer, for identificationand/or quantitation of the precursor ions in the MS1 domain; andperforming a set of MS2 scans, the set of MS2 scans comprising:segmenting the precursor ions into a plurality of precursor masssegments, each precursor mass segment having a mass range of no greaterthan 5 amu; wherein for each precursor mass segment: fragmenting theprecursor ions within that precursor mass segment; and performing an MS2scan of the fragmented ions by: forming an ion packet from thefragmented ions; and injecting the ion packet of fragmented ions into atime of flight mass analyzer and mass analyzing the fragmented ions. 2.A data independent acquisition method of mass spectrometry according toclaim 1 wherein: the Fourier Transform mass analyzer performs the MS1scans with a resolution of at least 50,000 at m/z=200; and/or theFourier Transform mass analyzer performs the MS1 scans with a massaccuracy of less 3 ppm.
 3. A data independent acquisition method of massspectrometry according to claim 1 wherein: the time of flight massanalyzer performs the MS2 scans with a resolution of at least 40,000 atm/z=200; and/or the time of flight mass analyzer performs the MS2 scanswith a mass accuracy of less than 5 ppm; and/or the time of flight massanalyzer performs the MS2 scans at a frequency of at least 100 Hz.
 4. Adata independent acquisition method of mass spectrometry according toclaim 1 wherein: each precursor mass segment has a mass range of nogreater than 3 amu.
 5. A data independent acquisition method of massspectrometry according to claim 1 wherein: the Fourier Transform massanalyzer is an orbital trapping mass analyzer.
 6. A data independentacquisition method of mass spectrometry according to claim 1 wherein: afragmentation energy for fragmenting each precursor mass sub-rangesegment is adjusted according to the mass range of each precursor masssub-range segment.
 7. A data independent acquisition method of massspectrometry according to claim 1 wherein: the ion packet of fragmentedions is formed by accumulating fragmented ions in an ion trap for apredetermined time.
 8. A data independent acquisition method of massspectrometry according to claim 1 wherein: at least 50% of the MS2 scanscontain more than 1000 ion counts.
 9. A data independent acquisitionmethod of mass spectrometry according to claim 1 wherein: performing theat least one MS1 scan of the precursor ions across the mass range ofinterest further comprises: separating the precursor ions in the massrange of interest into a plurality of mass sub-ranges, and for each masssub-range of precursor ions: mass analyzing the sub-range of precursorions using the Fourier Transform mass analyzer.
 10. A data independentacquisition method of mass spectrometry according to claim 1 wherein:the mass range of the precursor ions in each mass sub-range is nogreater than 300 amu, or more preferably 200 amu, or more preferably 100amu; and/or the mass range of interest is a range of at least 600 amu,or more preferably at least 800 amu or more preferably at least 1000amu.
 11. A data independent acquisition method of mass spectrometryaccording to claim 1 wherein: performing an MS2 scan of the set of MS2scans includes combining fragmented ions from a plurality of precursormass segments such that the combined fragmented ions are mass analysed.12. A data independent acquisition method of mass spectrometry accordingto claim 1 wherein: the MS1 scan and the set of MS2 scans are repeatedat least 4 times over the duration of the chromatographic peak.
 13. Adata independent acquisition method of mass spectrometry according toclaim 1 wherein: the MS1 scan and the set of MS2 scans are performed intandem.
 14. A mass spectrometer for performing data independent massspectrometry on a sample across a mass range of interest, the massspectrometer comprising: a chromatography system configured to separatemolecules of a sample; an ionisation source for producing a plurality ofprecursor ions from the sample molecules provided by the chromatographysystem; a mass selector; a Fourier Transform mass analyzer; afragmentation apparatus; an ion packet forming apparatus; a time offlight mass analyzer; a controller configured: i) to cause the massselector to select precursor ions within the mass range interest; andii) to cause the Fourier Transform mass analyzer to perform at least oneMS1 scan of the selected precursor ions for identification and/orquantitation of the precursor ions in the MS1 domain; and iii) to causethe time of flight mass analyzer to perform a set of MS2 scans wherein:the controller causes the mass selector to further segment the precursorions into a plurality of precursor mass segments, each precursor masssegment having a mass range of no greater than 5 amu; where for eachprecursor mass segment the controller causes: the fragmentationapparatus to fragment precursor ions within the precursor mass segmentinto a plurality of fragmented ions; the ion packet forming apparatus toform an ion packet from the fragmented ions; the ion packet formingapparatus to inject the fragmented ions into the time of flight massanalyzer; and the time of flight mass analyzer to mass analyse thefragmented ions across the mass range of interest.
 15. A massspectrometer according to claim 14 wherein: the Fourier Transform massanalyzer is configured to perform the MS1 scans with a resolution of atleast 50,000 at m/z=200; and/or the Fourier Transform mass analyzer isconfigured to perform the MS1 scans with a mass accuracy of less 3 ppm.16. A mass spectrometer according to claim 14 wherein: the time offlight mass analyzer is configured to perform the MS2 scans with aresolution of at least 40,000 at m/z=200; and/or the time of flight massanalyzer is configured to perform the MS2 scans with a mass accuracy ofless than 5 ppm; and/or the time of flight mass analyzer is configuredto perform the MS2 scans at a frequency of at least 100 Hz.
 17. A massspectrometer according to claim 14 wherein: the ion packet formingapparatus is a first ion trap.
 18. A mass spectrometer according toclaim 14 wherein the mass spectrometer further comprises: a branchedpath ion guide configured to guide precursor ions from the mass selectorto the Fourier transform mass analyzer and/or the fragmentation chamber.19. A mass spectrometer according to claim 18 further comprising: asecond ion trap, the second ion trap configured to trap precursor ionsprovided from the mass selector to the Fourier Transform mass analyzerand supply the ions to the Fourier Transform mass analyzer for the MS1scan; wherein the controller is configured to cause the second ion trapto trap precursor ions at a plurality of different times over theduration of the chromatographic peak, before supplying the ions to theFourier transform mass analyzer.
 20. A mass spectrometer according toclaim 14 wherein: the time of flight mass analyzer is a multiplereflection time of flight mass analyser.
 21. A mass spectrometeraccording to claim 14 wherein: the controller is configured to cause ionpackets formed by the ion packet forming apparatus provide that at least30% of the MS2 scans contain more than 200 ion counts.
 22. A massspectrometer according to claim 14 wherein: for the at least one MS1scans, the controller is configured: to cause the mass selector toselect precursor ions in a one of a plurality mass sub-ranges of themass range of interest; wherein for each mass sub-range of precursorions making up the mass range of interest: the controller is configuredto cause the Fourier Transform mass analyzer to mass analyzing the masssub-range of precursor ions.